This research focuses on the Human Immunodeficiency Virus type 1 RNase H activity of reverse transcriptase. RNase H activity is essential for the correct replication of the viral RNA to double-stranded DNA. Several of the RNase H cleavages show defined specificity which distinguish the viral enzyme from cellular proteins. This research proposal studies the unique cleavage associated with the specific removal of the tRNA(Lys,3) primer. Understanding the determinants which govern this specificity can ultimately lead to the development of specific inhibitors of the viral RNase H. Replication of a retrovirus is a complex reaction; the tRNA primer is removed at an intermediate step during DNA synthesis. Cleavage results the 3' terminal rA of the tRNA(Lys,3) remaining attached to the viral DNA. This position of the cleavage of the tRNA is unique to HIV-1 RNase H. This infers a specific sequence or structural recognition by the viral enzyme which may provide a mechanism of distinguishing the viral from host proteins. The experiments described aim at defining the determinants within the protein and the substrate for the specificity observed in the tRNA removal. An in vitro assay for tRNA removal has been developed which uses oligonucleotide substrates that mimics the replication intermediate. Studies aimed at defining the minimal substrate with respect to the RNA and DNA are described. The effect of mismatches and base substitutions will be determined. Footprint analysis of the tRNA/DNA substrate will be performed in order to pinpoint the specific region recognized by the protein. The protein determinants for the tRNA removal is intrinsic to the RNase H domain. The HIV-1 RNase H domain in the absence of the DNA polymerase domain has been expressed and is active. This isolated RNase H domain maintains the specificity for tRNA removal. Regions of the isolated RNase H domain which are in contact with the tRNA/DNA substrate will be determined using chemical crosslinking and artificial proteolysis. Kinetic analysis will determine the affinity of the isolated domain for various substrates. The removal of the tRNA exposes the sequences necessary for the second strand transfer event. However, little is known about the mechanism of release of the tRNA. The extent of degradation of the tRNA by the RNase H/reverse transcriptase will be determined using a kinetic analysis of the second strand transfer reaction. The requirement for the 3'->5' exonuclease of RNase H for the degradation of the tRNA will be examined.